CN108641013B - Preparation method of amidated pectin - Google Patents
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Abstract
The invention relates to a preparation method of amidated pectin, which comprises the steps of preparing clear pectin liquid from dried and crushed orange peels by adopting a mild extraction method, carrying out enzymolysis on the clear pectin liquid by adopting plant pectin methylesterase to finally obtain powdery initial pectin, and carrying out amidation reaction on the powdery initial pectin to obtain the amidated pectin, wherein the obtained amidated pectin has high molecular weight and a controllable block structure and meets the quality requirement standard of European Union (EU) on the amidated pectin. When the yoghurt is applied to yoghurt, the consistency of the yoghurt can be obviously increased, and the yoghurt does not have the phenomena of consistency reduction and bleeding after being placed for a long time. In addition, the amidated pectin prepared by the method can obviously change intestinal flora and promote the growth of intestinal probiotics.
Description
Technical Field
The invention relates to a preparation method of amidated pectin applied to yoghourt.
Background
Commercial pectin is a polysaccharide compound extracted from plant cell walls and is widely used in the food, health care and pharmaceutical industries as a food additive. Pectin is mainly composed of galacturonic acid, wherein part of galacturonic acid carboxyl groups are subjected to methyl esterification, and the esterification degree is 65-80%. To change the functionality of pectin, chemical de-esterification with acids, bases or ammonia can be used. Chemical de-esterification of pectin is a random process, and beta-degradation can occur to cause the cleavage of alpha-1, 4 glycosidic bonds between pectin and galacturonic acid, thereby reducing the molecular weight of the pectin. The molecular weight of pectin is very important for the functional properties of pectin, such as gelling, water retention, etc. Compared with a chemical method for degrading DE, the method has wide attraction and prospect by adopting an enzyme method. Although enzymatic de-esterification can reduce pectin degradation, plant methylesterase is rarely used and can be de-esterified to produce pectin with a controlled block structure while ensuring the pectin molecular weight. During the amidation reaction of pectin, the pectin undergoes changes in molecular weight and molecular conformation. The most significant factor affecting the molecular weight of pectin is the β -degradation reaction that occurs during the amidation reaction. The beta-degradation reaction will lead to a break of the pectin chains, thereby reducing the molecular weight and hence the viscosity of the pectin. The beta-degradation reaction is not only related to the temperature of the amidation reaction and the concentration of the alcohol ammonia solution, but also related to the initial pectin quality before the amidation reaction. The molecular weight of the initial pectin is related to the high thickening, high water retention effect the final amidated pectin plays in yoghurt. Furthermore, the DE distribution of the initial pectin is also related to the viscosity of the final amidated pectin in the yoghurt, and the presence of a block distribution of pectin non-methyl esterified galacturonic acid may increase the yoghurt viscosity. Thus, the combination of the degree of esterification and the molecular weight of the amidated initial pectin provides novel structural and functional properties of pectin, which can be applied in new products, health care, medicine and other applications.
Disclosure of Invention
The invention discloses a preparation method of amidated pectin, which adopts the following technical scheme:
1) preparation of Alcohol Insoluble Substance (AIS): taking the dried and crushed orange peels, adding 90% (v/v) alcohol with the volume being 3-4 times that of the orange peels, and slowly stirring for 10-20 min at 90 ℃; removing alcohol soluble substance containing small molecular sugar and pigment by centrifugation, oven drying the alcohol insoluble substance at 60 deg.C for 6 hr, and pulverizing;
2) preparation of a primary suspension: weighing the alcohol insoluble substance AIS, adding the AIS into deionized water at the temperature of 40-60 ℃, adjusting the mass ratio of the AIS to the deionized water to be 1: 10-1: 30, adjusting the pH to be 2.0-2.5 by using concentrated nitric acid, and stirring and extracting for 1-3 hours at the temperature of 40-60 ℃ and the rotating speed of 400rpm to obtain a primary extraction suspension;
3) preparing pectin clear liquid: filtering the primary extraction suspension obtained in the step 2) by using a vacuum rotary drum filter to obtain filtrate, namely pectin clear liquid;
4) enzymolysis: adding the clear liquid obtained in the step 3) into a stainless steel heat-preservation tank, maintaining the temperature at 45-55 ℃, adjusting the pH value of enzymolysis to 4.0-4.5 by adopting a 10% sodium carbonate solution, adding pectin methylesterase with the mass of 20-50 ppm of the concentrated solution into the clear liquid, starting stirring, reacting at the rotating speed of 100rpm, and stopping the reaction when the esterification degree of the clear liquid reaches the required esterification degree;
5) enzyme deactivation: inactivating enzyme of the clear liquid obtained in the step 4) for 10-15 min at the temperature of 75-85 ℃ and the pH of 2.0-2.5, and then rapidly cooling to below 50 ℃;
6) and (3) ultrafiltration: carrying out ultrafiltration on the clear liquid after enzyme deactivation in the step 5) by adopting an ultrafiltration membrane with the interception relative molecular weight of 1000u, adding deionized water, washing and concentrating to the original volume of 1/3-1/4;
7) preparing a washing filter cake: adding 70% ethanol into the concentrated solution obtained in the step 6), wherein the volume ratio of the concentrated solution to the 70% ethanol is 1:3, filtering to obtain a filter cake, washing for 3-5 times by using the 70% ethanol, wherein the mass ratio of the 70% ethanol to the filter cake used in each washing is 5-10: 1, and stirring for 30min to obtain a washing filter cake;
8) preparation of powdered primary pectin: drying the washing filter cake obtained in the step 7) at 60 ℃, crushing and screening after drying to obtain powdery initial pectin;
9) preparation of an alcoholic ammonia solution: mixing concentrated ammonia water with 70% ethanol to obtain an ammonia-alcohol solution, wherein the ammonia concentration of the ammonia-alcohol solution is 3% -5%;
10) preparation of amidated pectin suspensions: mixing the alcohol ammonia solution prepared in the step 9) and the powder filter cake prepared in the step 8) according to the ratio of 4:1(v/w), and stirring for 3-6h at the temperature of 6 ℃ and the rotating speed of 100r/min to obtain amidated pectin suspension;
11) preparing amidated pectin for yoghourt: filtering the amidated pectin suspension obtained in the step 10) to obtain a primary filter cake, adding 60% ethanol into the primary filter cake, wherein the w/v ratio of the primary filter cake to the 60% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a secondary filter cake, adding 50-70% ethanol into the secondary filter cake, wherein the w/v ratio of the secondary filter cake to the 50-70% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a tertiary filter cake, adding 60% ethanol into the tertiary filter cake, wherein the w/v ratio of the tertiary filter cake to the 60% ethanol is 1:2, adding 62-65% w/w concentrated nitric acid, adjusting the pH value to 4.2 for four times, filtering to obtain a filter cake, drying the quaternary filter cake to a water content of less than or equal to 10%, crushing, and screening to obtain the amidated pectin applied to the yogurt;
the mass concentration of the concentrated nitric acid is 62-65%;
the pectin methylesterase is separated from orange peel, and can be de-esterified to generate controllable pectin methylesterase with a block structure.
And 8) the mesh number of the initial pectin of the powder is 50-150 meshes, and the average particle size is 100 meshes.
The invention has the following beneficial technical effects:
1. the adoption of plant pectin methylesterase can prepare amidated pectin with high molecular weight and controllable block structure, and has mild operation condition and less environmental pollution.
2. The method selects the initial pectin with the molecular weight of more than 150000 and DE of 30-35 percent prepared by the pectin methyl ester enzyme method, and the amidated pectin generated by amidation reaction is applied to the yoghourt, has the application characteristics of obvious thickening, low water drainage rate and the like, has uniform structure and fine texture, and does not cause the phenomena of thickness reduction and water drainage after being placed for a long time.
3. The pectin methylesterase is separated from orange peel, and can be de-esterified to generate controllable pectin methylesterase with a block structure; plant pectin methylesterase attacks and removes methoxyl from the reducing end of pectin molecule or the adjacent free carboxyl along the single chain of the molecule to form de-esterified pectin with a partition block structure.
4. Initial pectin with the molecular weight of more than 150000 and DE of 30-35 percent prepared by a pectin methyl ester enzyme method is selected, and amidated pectin generated by amidation reaction can obviously change intestinal flora and promote the growth of intestinal probiotics.
Detailed Description
Example 1:
1) preparation of Alcohol Insoluble Substance (AIS): adding 90% (v/v) ethanol 3 times the volume of the dried and pulverized pericarpium Citri Junoris, and slowly stirring at 90 deg.C for 20 min. Removing alcohol soluble substance containing small molecular sugar and pigment by centrifugation, oven drying the alcohol insoluble substance at 60 deg.C for 6 hr, and pulverizing.
2) Preparation of a primary suspension: weighing the alcohol insoluble substance AIS, adding the AIS and deionized water into deionized water at 50 ℃, adjusting the mass ratio of AIS to deionized water to be 1:20, adjusting the pH to be 2.5 by using concentrated nitric acid, and stirring and extracting for 2 hours under the conditions that the temperature is 50 ℃ and the rotating speed is 400rpm to obtain a primary extract suspension;
3) preparing pectin clear liquid: filtering the primary extraction suspension obtained in the step 2) by using a vacuum rotary drum filter to obtain filtrate, namely pectin clear liquid;
4) enzymolysis: adding the clear liquid obtained in the step 3) into a stainless steel heat-preservation tank, keeping the temperature at 50 ℃, adjusting the pH value of enzymolysis by adopting a 10% sodium carbonate solution, adding pectin methylesterase with the mass of 30ppm of the concentrated solution into the clear liquid, starting stirring, reacting at the rotating speed of 100rpm, and stopping the reaction when the esterification degree of the clear liquid reaches the required esterification degree;
5) enzyme deactivation: inactivating enzyme of the clear liquid obtained in the step 4) for 10-15 min at the temperature of 80 ℃ and the pH value of 2.0-2.5, and then rapidly cooling to below 50 ℃;
6) and (3) ultrafiltration: and (3) carrying out ultrafiltration on the clear liquid after enzyme deactivation in the step 5) by adopting an ultrafiltration membrane with the interception relative molecular weight of 1000u, adding deionized water, washing and concentrating to the original volume of 1/4.
7) Preparing a washing filter cake: adding 70% ethanol into the concentrated solution obtained in the step 6), wherein the volume ratio of the concentrated solution to the 70% ethanol is 1:3, filtering to obtain a filter cake, washing for 5 times by using the 70% ethanol, wherein the mass ratio of the 70% ethanol to the filter cake used in each washing is 10:1, and stirring for 30min to obtain a washing filter cake;
8) preparation of powdered primary pectin: drying the washing filter cake obtained in the step 7) at 60 ℃, crushing and screening after drying to obtain powdery initial pectin;
| initial pectin | DE | Molecular weight (Mw) |
| 51.5 | 144750 |
The results are shown in the table.
9) Preparation of an alcoholic ammonia solution: mixing concentrated ammonia water with 70% ethanol to obtain an ammonia-alcohol solution, wherein the ammonia concentration of the ammonia-alcohol solution is 5%;
10) preparation of amidated pectin suspensions: mixing the alcohol ammonia solution prepared in the step 9) and the powder filter cake prepared in the step 8) according to the ratio of 4:1(v/w), and stirring for 3-6h at the temperature of 6 ℃ and the rotating speed of 100r/min to obtain amidated pectin suspension;
11) preparing amidated pectin for yoghourt: filtering the amidated pectin suspension obtained in the step 10) to obtain a primary filter cake, adding 60% ethanol into the primary filter cake, wherein the w/v ratio of the primary filter cake to the 60% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a secondary filter cake, adding 50-70% ethanol into the secondary filter cake, the w/v ratio of the secondary filter cake to the 50-70% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a tertiary filter cake, adding 60% ethanol into the tertiary filter cake, wherein the w/v ratio of the tertiary filter cake to the 60% ethanol is 1:2, adding 62-65% w/w concentrated nitric acid, adjusting the pH value to 4.2 for four times, filtering to obtain a filter cake, drying the fourth filter cake to the water content of less than or equal to 10%, crushing, and screening to obtain the amidated pectin;
| amidated pectin | DE (original) | DA (Final) | DE (Final) | Molecular weight (Mw) |
| 51.5 | 21.3 | 23.2 | 140860 |
The results are shown in the table.
Example 2:
1) preparation of Alcohol Insoluble Substance (AIS): adding 90% (v/v) ethanol 3 times the volume of the dried and pulverized pericarpium Citri Junoris, and slowly stirring at 90 deg.C for 20 min. Removing alcohol soluble substance containing small molecular sugar and pigment by centrifugation, oven drying the alcohol insoluble substance at 60 deg.C for 6 hr, and pulverizing.
2) Preparation of a primary suspension: weighing the alcohol insoluble substance AIS, adding the AIS and deionized water into deionized water at 50 ℃, adjusting the mass ratio of AIS to deionized water to be 1:20, adjusting the pH to be 2.5 by using concentrated nitric acid, and stirring and extracting for 2 hours under the conditions that the temperature is 50 ℃ and the rotating speed is 400rpm to obtain a primary extract suspension;
3) preparing pectin clear liquid: filtering the primary extraction suspension obtained in the step 2) by using a vacuum rotary drum filter to obtain filtrate, namely pectin clear liquid;
4) enzymolysis: adding the clear liquid obtained in the step 3) into a stainless steel heat-preservation tank, keeping the temperature at 50 ℃, adjusting the pH value of enzymolysis by adopting a 10% sodium carbonate solution, adding pectin methylesterase with the mass of 36ppm of the concentrated solution into the clear liquid, starting stirring, reacting at the rotating speed of 100rpm, and stopping the reaction when the esterification degree of the clear liquid reaches the required esterification degree;
5) enzyme deactivation: inactivating enzyme of the clear liquid obtained in the step 4) for 10-15 min at the temperature of 80 ℃ and the pH value of 2.0-2.5, and then rapidly cooling to below 50 ℃;
6) and (3) ultrafiltration: and (3) carrying out ultrafiltration on the clear liquid after enzyme deactivation in the step 5) by adopting an ultrafiltration membrane with the interception relative molecular weight of 1000u, adding deionized water, washing and concentrating to the original volume of 1/4.
7) Preparing a washing filter cake: adding 70% ethanol into the concentrated solution obtained in the step 6), wherein the volume ratio of the concentrated solution to the 70% ethanol is 1:3, filtering to obtain a filter cake, washing for 5 times by using the 70% ethanol, wherein the mass ratio of the 70% ethanol to the filter cake used in each washing is 10:1, and stirring for 30min to obtain a washing filter cake;
8) preparation of powdered primary pectin: drying the washing filter cake obtained in the step 7) at 60 ℃, crushing and screening after drying to obtain powdery initial pectin;
| initial pectin | DE | Molecular weight (Mw) |
| 43.5 | 133900 |
The results are shown in the table.
9) Preparation of an alcoholic ammonia solution: mixing concentrated ammonia water with 70% ethanol to obtain an ammonia-alcohol solution, wherein the ammonia concentration in the ammonia-alcohol solution is 4.5%;
10) preparation of amidated pectin suspensions: mixing the alcohol ammonia solution prepared in the step 9) and the powder filter cake prepared in the step 8) according to the ratio of 4:1(v/w), and stirring for 3-6h at the temperature of 6 ℃ and the rotating speed of 100r/min to obtain amidated pectin suspension;
11) preparing amidated pectin for yoghourt: filtering the amidated pectin suspension obtained in the step 10) to obtain a primary filter cake, adding 60% ethanol into the primary filter cake, wherein the w/v ratio of the primary filter cake to the 60% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a secondary filter cake, adding 50-70% ethanol into the secondary filter cake, the w/v ratio of the secondary filter cake to the 50-70% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a tertiary filter cake, adding 60% ethanol into the tertiary filter cake, wherein the w/v ratio of the tertiary filter cake to the 60% ethanol is 1:2, adding 62-65% w/w concentrated nitric acid, adjusting the pH value to 4.2 for four times, filtering to obtain a filter cake, drying the fourth filter cake to the water content of less than or equal to 10%, crushing, and screening to obtain the amidated pectin;
the results are shown in the table.
Example 3:
1) preparation of Alcohol Insoluble Substance (AIS): adding 90% (v/v) ethanol 3 times the volume of the dried and pulverized pericarpium Citri Junoris, and slowly stirring at 90 deg.C for 20 min. Removing alcohol soluble substance containing small molecular sugar and pigment by centrifugation, oven drying the alcohol insoluble substance at 60 deg.C for 6 hr, and pulverizing.
2) Preparation of a primary suspension: weighing the alcohol insoluble substance AIS, adding the AIS and deionized water into deionized water at 50 ℃, adjusting the mass ratio of AIS to deionized water to be 1:20, adjusting the pH to be 2.5 by using concentrated nitric acid, and stirring and extracting for 2 hours under the conditions that the temperature is 50 ℃ and the rotating speed is 400rpm to obtain a primary extract suspension;
3) preparing pectin clear liquid: filtering the primary extraction suspension obtained in the step 2) by using a vacuum rotary drum filter to obtain filtrate, namely pectin clear liquid;
4) enzymolysis: adding the clear liquid obtained in the step 3) into a stainless steel heat-preservation tank, keeping the temperature at 50 ℃, adjusting the pH value of enzymolysis by adopting a 10% sodium carbonate solution, adding pectin methylesterase with the mass of 36ppm of the concentrated solution into the clear liquid, starting stirring, reacting at the rotating speed of 100rpm, and stopping the reaction when the esterification degree of the clear liquid reaches the required esterification degree;
5) enzyme deactivation: inactivating enzyme of the clear liquid obtained in the step 4) for 10-15 min at the temperature of 80 ℃ and the pH value of 2.0-2.5, and then rapidly cooling to below 50 ℃;
6) and (3) ultrafiltration: and (3) carrying out ultrafiltration on the clear liquid after enzyme deactivation in the step 5) by adopting an ultrafiltration membrane with the interception relative molecular weight of 1000u, adding deionized water, washing and concentrating to the original volume of 1/4.
7) Preparing a washing filter cake: adding 70% ethanol into the concentrated solution obtained in the step 6), wherein the volume ratio of the concentrated solution to the 70% ethanol is 1:3, filtering to obtain a filter cake, washing for 5 times by using the 70% ethanol, wherein the mass ratio of the 70% ethanol to the filter cake used in each washing is 10:1, and stirring for 30min to obtain a washing filter cake;
8) preparation of powdered primary pectin: drying the washing filter cake obtained in the step 7) at 60 ℃, crushing and screening after drying to obtain powdery initial pectin;
| initial pectin | DE | Molecular weight (Mw) |
| 35.8 | 158900 |
The results are shown in the table.
9) Preparation of an alcoholic ammonia solution: mixing concentrated ammonia water with 70% ethanol to obtain an ammonia-alcohol solution, wherein the ammonia concentration in the ammonia-alcohol solution is 4.5%;
10) preparation of amidated pectin suspensions: mixing the alcohol ammonia solution prepared in the step 9) and the powder filter cake prepared in the step 8) according to the ratio of 4:1(v/w), and stirring for 3-6h at the temperature of 6 ℃ and the rotating speed of 100r/min to obtain amidated pectin suspension;
11) preparing amidated pectin for yoghourt: filtering the amidated pectin suspension obtained in the step 10) to obtain a primary filter cake, adding 60% ethanol into the primary filter cake, wherein the w/v ratio of the primary filter cake to the 60% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a secondary filter cake, adding 50-70% ethanol into the secondary filter cake, the w/v ratio of the secondary filter cake to the 50-70% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a tertiary filter cake, adding 60% ethanol into the tertiary filter cake, wherein the w/v ratio of the tertiary filter cake to the 60% ethanol is 1:2, adding 62-65% w/w concentrated nitric acid, adjusting the pH value to 4.2 for four times, filtering to obtain a filter cake, drying the fourth filter cake to the water content of less than or equal to 10%, crushing, and screening to obtain the amidated pectin;
| amidated pectin | DE (original) | DA (Final) | DE (Final) | Molecular weight (Mw) |
| 35.8 | 17.5 | 13.9 | 160060 |
The results are shown in the table.
Example 4:
1) preparation of Alcohol Insoluble Substance (AIS): adding 90% (v/v) ethanol 3 times the volume of the dried and pulverized pericarpium Citri Junoris, and slowly stirring at 90 deg.C for 20 min. Removing alcohol soluble substance containing small molecular sugar and pigment by centrifugation, oven drying the alcohol insoluble substance at 60 deg.C for 6 hr, and pulverizing.
2) Preparation of a primary suspension: weighing the alcohol insoluble substance AIS, adding the AIS and deionized water into deionized water at 50 ℃, adjusting the mass ratio of AIS to deionized water to be 1:20, adjusting the pH to be 2.5 by using concentrated nitric acid, and stirring and extracting for 2 hours under the conditions that the temperature is 50 ℃ and the rotating speed is 400rpm to obtain a primary extract suspension;
3) preparing pectin clear liquid: filtering the primary extraction suspension obtained in the step 2) by using a vacuum rotary drum filter to obtain filtrate, namely pectin clear liquid;
4) enzymolysis: adding the clear liquid obtained in the step 3) into a stainless steel heat-preservation tank, keeping the temperature at 50 ℃, adjusting the pH value of enzymolysis by adopting a 10% sodium carbonate solution, adding pectin methylesterase with the mass of 36ppm of the concentrated solution into the clear liquid, starting stirring, reacting at the rotating speed of 100rpm, and stopping the reaction when the esterification degree of the clear liquid reaches the required esterification degree;
5) enzyme deactivation: inactivating enzyme of the clear liquid obtained in the step 4) for 10-15 min at the temperature of 80 ℃ and the pH value of 2.0-2.5, and then rapidly cooling to below 50 ℃;
6) and (3) ultrafiltration: and (3) carrying out ultrafiltration on the clear liquid after enzyme deactivation in the step 5) by adopting an ultrafiltration membrane with the interception relative molecular weight of 1000u, adding deionized water, washing and concentrating to the original volume of 1/4.
7) Preparing a washing filter cake: adding 70% ethanol into the concentrated solution obtained in the step 6), wherein the volume ratio of the concentrated solution to the 70% ethanol is 1:3, filtering to obtain a filter cake, washing for 5 times by using the 70% ethanol, wherein the mass ratio of the 70% ethanol to the filter cake used in each washing is 10:1, and stirring for 30min to obtain a washing filter cake;
8) preparation of powdered primary pectin: drying the washing filter cake obtained in the step 7) at 60 ℃, crushing and screening after drying to obtain powdery initial pectin;
| initial pectin | DE | Molecular weight (Mw) |
| 29.9 | 153500 |
The results are shown in the table.
9) Preparation of an alcoholic ammonia solution: mixing concentrated ammonia water with 70% ethanol to obtain an ammonia-alcohol solution, wherein the ammonia concentration in the ammonia-alcohol solution is 4.5%;
10) preparation of amidated pectin suspensions: mixing the alcohol ammonia solution prepared in the step 9) and the powder filter cake prepared in the step 8) according to the ratio of 4:1(v/w), and stirring for 3-6h at the temperature of 6 ℃ and the rotating speed of 100r/min to obtain amidated pectin suspension;
11) preparing amidated pectin for yoghourt: filtering the amidated pectin suspension obtained in the step 10) to obtain a primary filter cake, adding 60% ethanol into the primary filter cake, wherein the w/v ratio of the primary filter cake to the 60% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a secondary filter cake, adding 50-70% ethanol into the secondary filter cake, the w/v ratio of the secondary filter cake to the 50-70% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a tertiary filter cake, adding 60% ethanol into the tertiary filter cake, wherein the w/v ratio of the tertiary filter cake to the 60% ethanol is 1:2, adding 62-65% w/w concentrated nitric acid, adjusting the pH value to 4.2 for four times, filtering to obtain a filter cake, drying the fourth filter cake to the water content of less than or equal to 10%, crushing, and screening to obtain the amidated pectin;
| amidated pectin | DE (original) | DA (Final) | DE (Final) | Molecular weight (Mw) |
| 29.9 | 14.8 | 13.9 | 155060 |
The results are shown in the table.
The beneficial effects of the present invention are further illustrated below in conjunction with experimental data:
experiment one:
1, materials and methods:
1.1 test site: laboratory of product development center of Nicoti Andeli pectin GmbH
1.2 subjects: amidated pectins prepared according to the invention (example 3), comparative 1 (amidated pectin prepared in example 1) and comparative 2 (amidated pectin prepared in patent ZL 200510021986.1).
1.3 detection method:
detecting the Degree of Esterification (DE) and Degree of Amidation (DA) by QB2484-2000 food additive pectin detection method, and determining pectin molecular weight by high performance liquid chromatography.
Except for different treatments, the conditions of the experiment are consistent.
2 results and analysis:
TABLE 1
As can be seen from a comparison of the present invention and comparative example 1 in Table 1, the initial pectin prepared by the enzymatic method has no significant effect on the molecular weight of the amidated pectin produced by the subsequent amidation reaction due to the different DE of the initial pectin. Compared with the comparison 1 and the comparison 2, the molecular weight of the amidated pectin finally obtained by the enzyme method is obviously higher than that of the amidated pectin obtained by the acid method.
The 3 kinds of amidated pectin are used in producing sour milk, the pectin consumption is 0.1-0.2%, the protein content in the product is 3-4%, the consistency and water separating rate of the product are detected, and the phenomenon is observed, which is shown in Table 2
TABLE 2
Compared with the data, the invention is applied to the yoghourt, has the application characteristics of obvious thickening, low bleeding rate and the like, has uniform structure and fine texture, and does not generate the phenomena of thickness reduction and bleeding after being placed for a long time.
Experiment two:
1, materials and methods:
1.1 test site: nicoti Andeli pectin, Inc. product development center laboratories.
1.2 subjects: amidated pectins prepared according to the invention (example 3), comparative 1 (amidated pectin prepared in example 1) and comparative 2 (amidated pectin prepared in patent ZL 200510021986.1), comparative 3 (bifidobacterium bifidum Bifico).
1.3 detection method:
40 cases of colitis patients are selected, 10 cases of health examiners are taken as a normal group, stool samples are collected, intestinal flora is measured, and the change of the intestinal flora composition of the patients among different groups is observed. Randomly dividing 40 patients with colitis into 5 groups, and 10 patients with colitis in control group without taking pectin and probiotics; the other 3 groups were administered with 5 g/dose, 2/dose of each of amidated pectin prepared according to the present invention (example 3), amidated pectin prepared according to example 1 (comparative 1) and amidated pectin prepared according to patent ZL200510021986.1 (comparative 2); the fifth group took 2 granules of Bifidobacterium triple viable Bifico (comparison 3)2 times/d. The total treatment course is 4 weeks, and fecal specimens before and after administration are collected respectively for detection.
The experiment was carried out under otherwise identical conditions except for the materials.
2 results and analysis:
the colitis patients without any pectin or probiotics have intestinal flora disorder, compared with the normal group, the quantity of bifidobacteria and lactobacilli in the excrement of the colitis patients is obviously reduced, the quantity of escherichia coli is obviously increased, and the difference has statistical significance (P is less than 0.05).
After the administration of the amidated pectin prepared according to the present invention, the amidated pectin prepared in example 1 and the viable bifidobacterium triple bifidum Bifico, the number of bifidobacteria and lactobacilli in the feces of the patients increased, while the number of coliform bacteria decreased, and the differences were statistically significant (P < 0.05). Compared with the amidated pectin prepared in the example 1 and the bifidobacterium triple viable Bifico, the number of the bifidobacteria and the lactobacilli in the excrement of the patient is obviously increased and the number of escherichia coli is obviously reduced (P is less than 0.05) after the amidated pectin prepared by the invention is taken.
However, after the amidated pectin prepared in patent ZL200510021986.1 is taken, the number of bifidobacteria and lactobacilli in the excrement of the patient is slightly increased, and the remarkable difference exists in comparison with the invention, comparison 1 and comparison 3.
And (4) conclusion: by taking the amidated pectin prepared by the invention, the intestinal flora can be obviously changed, and the growth of intestinal probiotics is promoted.
Claims (2)
1. A preparation method of amidated pectin is characterized in that: selecting pectin methyl ester enzyme method to prepare initial pectin with molecular weight of more than 150000 and DE =30-35%, amidated pectin generated by amidation reaction, and specifically performing the following steps:
1) preparing alcohol insoluble substance AIS: taking the dried and crushed orange peels, adding 3-4 times of 90% alcohol by volume, slowly stirring for 10-20 min at 90 ℃, removing alcohol soluble substances containing small molecular sugar and pigments through centrifugation, drying alcohol insoluble substances for 6h at 60 ℃, and crushing for later use;
2) preparation of a primary suspension: weighing the alcohol insoluble substance AIS, adding the AIS into deionized water at the temperature of 40-60 ℃, adjusting the mass ratio of the AIS to the deionized water to be 1: 10-1: 30, adjusting the pH to be 2.0-2.5 by using concentrated nitric acid, and stirring and extracting for 1-3 hours at the temperature of 40-60 ℃ and the rotating speed of 400rpm to obtain a primary extraction suspension;
3) preparing pectin clear liquid: filtering the primary extraction suspension obtained in the step 2) by using a vacuum rotary drum filter to obtain filtrate, namely pectin clear liquid;
4) enzymolysis: adding the clear liquid obtained in the step 3) into a stainless steel heat-preservation tank, maintaining the temperature at 45-55 ℃, adjusting the pH value of enzymolysis to 4.0-4.5 by adopting a 10% sodium carbonate solution, adding pectin methylesterase with the mass of 20-50 ppm of the concentrated solution into the clear liquid, starting stirring, reacting at the rotating speed of 100rpm, and stopping the reaction when the esterification degree of the clear liquid reaches the required esterification degree;
5) enzyme deactivation: inactivating enzyme of the clear liquid obtained in the step 4) for 10-15 min at the temperature of 75-85 ℃ and the pH of 2.0-2.5, and then rapidly cooling to below 50 ℃;
6) and (3) ultrafiltration: carrying out ultrafiltration on the clear liquid after enzyme deactivation in the step 5) by adopting an ultrafiltration membrane with the interception relative molecular weight of 1000u, adding deionized water, washing and concentrating to the original volume of 1/3-1/4;
7) preparing a washing filter cake: adding 70% ethanol into the concentrated solution obtained in the step 6), wherein the volume ratio of the concentrated solution to the 70% ethanol is 1:3, filtering to obtain a filter cake, washing for 3-5 times by using the 70% ethanol, wherein the mass ratio of the 70% ethanol to the filter cake used in each washing is 5-10: 1, and stirring for 30min to obtain a washing filter cake;
8) preparation of powdered primary pectin: drying the washing filter cake obtained in the step 7) at 60 ℃, crushing and screening after drying to obtain powdery initial pectin;
9) preparation of an alcoholic ammonia solution: mixing concentrated ammonia water with 70% ethanol to obtain an ammonia-alcohol solution, wherein the ammonia concentration of the ammonia-alcohol solution is 3% -5%;
10) preparation of amidated pectin suspensions: mixing the alcohol ammonia solution prepared in the step 9) and the powder filter cake prepared in the step 8) according to the volume mass ratio of 4:1, and stirring for 3-6h at the temperature of 6 ℃ and the rotating speed of 100r/min to obtain amidated pectin suspension;
11) preparing amidated pectin for yoghourt: filtering the amidated pectin suspension obtained in the step 10) to obtain a primary filter cake, adding 60% ethanol into the primary filter cake, wherein the mass-to-volume ratio of the primary filter cake to the 60% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a secondary filter cake, adding 50-70% ethanol into the secondary filter cake, wherein the mass-to-volume ratio of the secondary filter cake to the 50-70% ethanol is 1:2, stirring at 60rpm for 30min, filtering to obtain a tertiary filter cake, adding 60% ethanol into the tertiary filter cake, wherein the mass-to-volume ratio of the tertiary filter cake to the 60% ethanol is 1:2, adding 62-65% w/w concentrated nitric acid, adjusting the pH to 4.2, filtering to obtain a quaternary filter cake, drying the quaternary filter cake to a water content of less than or equal to 10%, crushing, and screening to obtain amidated pectin applied to yoghourt;
the mass concentration of the concentrated nitric acid is 62-65%;
the pectin methylesterase is separated from orange peel and can be subjected to deesterification to generate controllable pectin methylesterase with a block structure; plant pectin methylesterase attacks and removes methoxyl from the reducing end of pectin molecule or the adjacent free carboxyl along the single chain of the molecule to form de-esterified pectin with a partition block structure.
2. The method of claim 1, wherein the amidated pectin is prepared by: and 8) the mesh number of the initial pectin of the powder is 50-150 meshes, and the average particle size is 100 meshes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60161401A (en) * | 1984-01-31 | 1985-08-23 | Mitsui Toatsu Chem Inc | Separation method of pectin substrate |
| CN1786035A (en) * | 2005-11-02 | 2006-06-14 | 西华大学 | Production technology of amidated pectin |
| CN101817886A (en) * | 2009-12-18 | 2010-09-01 | 烟台安德利果胶有限公司 | Method for producing amidated pectin |
| CN105131148A (en) * | 2015-09-07 | 2015-12-09 | 三门峡康瑞药业有限公司 | Preparation method for amidated pectin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60161401A (en) * | 1984-01-31 | 1985-08-23 | Mitsui Toatsu Chem Inc | Separation method of pectin substrate |
| CN1786035A (en) * | 2005-11-02 | 2006-06-14 | 西华大学 | Production technology of amidated pectin |
| CN101817886A (en) * | 2009-12-18 | 2010-09-01 | 烟台安德利果胶有限公司 | Method for producing amidated pectin |
| CN105131148A (en) * | 2015-09-07 | 2015-12-09 | 三门峡康瑞药业有限公司 | Preparation method for amidated pectin |
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| Title |
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| 大豆皮果胶酶法脱脂参数研究;刘贺等;《食品工业科技》;20110531;第32卷(第5期);273-274 * |
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